The overall objective of the work proposed is to understand the function of clathrin coated vesicles and coated vesicle proteins in living cells. Dr. O'Halloran's focus has been to study the physiology and morphology of clathrin- depleted Dictyostelium cells. These mutants are viable, but they lack endosomes and a contractile vacuole, an organelle important for adaptation to osmotic stress. In addition, the cells exhibit severe deficiencies in endocytosis and osmoregulation. The purpose of this study is to dissect the contribution of clathrin to these physiological processes.She has two major goals. (1) The mechanism for clathrin's role in cellular function will be determined. Conditional mutants for clathrin heavy chain protein will be created and studied. The relationship between the absence of clathrin heavy chain protein, the rearrangements of contractile vacuole markers and the loss of osmoregulatory activity in mutant cells will be determined. The hypothesis that clathrin directly mediates fluid-phase and receptor-mediated endocytosis will be tested. The role of clathrin in the sorting, processing and targeting of lysosomal enzymes will be determined. (2) The intracellular role of clathrin light chains and adaptins, possible regulators of clathrin function will be determined. Coated vesicles from Dictyostelium will be isolate and the constituent proteins. cDNAs for clathrin light chains and the adaptin/assembly protein complex will be cloned from a Dictyostelium library. Mutants that contain deletions in these genes or which overexpress functional domains of the gene products will be constructed. The phenotype of these mutants will be studied in detail to test the hypothesis that these gene products function in the regulation of clathrin traffic.